Project Summary Genetic variants that hyperactivate mechanistic target of rapamycin (mTOR) signaling are among the most common pathological substrates associated with intractable pediatric epilepsy, and hyperactivation of the mTOR pathway is also proposed to mediate epileptogenesis in response to brain injury. Although altered neuronal migration and morphology are hallmarks of many known mTOR-related neurological diseases (mTORopathies) in humans, studies in animal models show that abnormal synaptic transmission and network activity precede or occur in the absence of overt structural changes, and that preventing structural changes does not prevent the neurological symptoms. This highlights the need for a better understanding of the functional changes in the brain. This proposal will test the hypothesis that abnormal synchronous neuronal activity caused by genetic hyperactivation of the mTOR signaling pathway is driven by changes in synaptic transmission. The long-term goal is to understand the genesis of, and then prevent or rescue, this abnormal activity, which may underlie both the high incidence of epilepsy and autism in humans with mTORopathies. In Aim 1, we will address this by testing four genetic models of mTORopathies (Tsc1, Pten, Pik3ca, Szt2) and determining whether there are common synaptic changes. Whether different mTORopathies share common synaptic alterations is an essential question to understanding the mechanistic similarity of these molecularly related diseases. In Aim 2, we will use molecular genetic rescue strategies that dissociate the morphological and synaptic effects of mTOR hyperactivation to test whether synaptic changes are sufficient to induce hypersynchronous activity and epilepsy. In Aim 3, we will use a combination of widefield and 2-photon calcium imaging to track the development and characteristics of hypersynchronous activity in vivo. We will then test whether the synaptic changes we observe in vitro are present at the time and place of hypersynchronous activity onset, and whether they can drive aberrant network activity. We anticipate that defining the functional consequences of mTOR hyperactivation relevant to enhanced neuronal excitability will lead to significant advances in the understanding of disease mechanisms, and aid the development of treatment strategies for mTORpathies and other neurological diseases.